Bill's Talk:
presented an ASM talk; Mentioned that he presents speeches over the world, updated in different places. Bill says MTB is the world's most susseccful path. Survival guide for pathogen, we'd be able to have perspectives from the worlds most succ one. Jacque rosee, leprosy research at Johns Hopkins, working on Tb there, I met him years ago...as grad student in 83, made first genomic lep DNA libraries...next slide is armadillo and leprosy result; Bill as handler of dasypus novansynctus = 9 banded armadillo. were 2 leper coloniesin US, 1 on Malachi Hawai..movie Fr. Demian. Simpsons leprosy agency... were 2 leper colonies in US, 1 outside New Orleans, other Malachi, HW. Patient in leper colony , went in , and shown 2 years later, very disfigured. Vrs. letting run the streets of NYC...story of being wisked away from families. leprosy still very much with us today, 5 mill world wide cases today.
leprosy organism only grows...for my thesis work 1 ...does not grow on media like smeg. instead, it grows in people, 1960 charles sheppard showed it would grow in foot pad of mouse, cooler part of body. 9 b armadillo; people lesions in face, hands = cooler part body. zoology text, animals w low body temp are hedghog and 9 b armadillo. cargil ? biopsies, New Orleans, leprosy patch into armadillo, doubles 1 every 14 days. e.coli every 20 min; smeg is 2,5-3 h. time to go from single cell to colony, e.coli is 8 h, smeg is 3-6 days. our d-29 r mutants took 5-6 days. MTB is once a day. 2 years to get a leprosy colony; so not grown. Biopsy--Armadillo, 2 years, then liver and spleen packed thru with bacillus.
since nothin is known about the genetics of m. leprae, we decided to make genomic libraries. --zhack rossea, cargill lousiana meeting, this line was bill's opening line at that talk. but at the meeting, i panicked, and what i said:
"sincing nothing is known about m. leprae, we decided to do genetics. I was ridiculed for that with the professionals at that meeting.
tb is a global problem in tuberculosis, 1 of 3 people on planet earth is subclinically infected with Tb. 2-3 million people die each year with Tb. Per year, it is 2-3 million. per day, comes out to 4700 to 8500 people that die each day of every year for tuberculosis. we've known relatively little about tb, we've known about BCG for many years...attenuated m. bovis (cattle), we've never knwon how it is attenuated, in a month, we have a paper that will explain how it is attenuated. we have used drugs...used over 40 years
[[I spoke to students during the talk for awhile.]]
Bill spoke about drugs in S.Africa, where they are available in the Black Market. Short Course chemo is minimum of 6 months treatment. Pills must be taken each month...people say I feel better, and I don't need to take the pills, but we need to continue taking the pills...if a patient stops taking the drugs, we have an emergence of drug resistant bacteria. 1 drug resistant infection, from 6 month treatment to minimum of 2 years. Compliance becomes worse. We ensure people in the us via DOTS, directly observed therapy. In NYC, we have nurses that ensure people take their pills each day. I.e., homeless people could be known by the nurse who ensures that pills are taken each day. Can you imagine the cost of this? But this is the most effective way to treat. With strep, 3 days feel better, 10 days and cured. Not so with Tb.
WHO has declared Tb a global health emergency, not done for any other disesase.
R. Koch , photo. He is one of our heros of Tb research. He is a german sci who discovered the Tb bacillus. he was the 1st person that actually proved a bacterium could cause a disease. noble prize, 1905 for discovery of Tb bacillus.
Edward Livingston Trudeau...who has seen the Doonsbury comics? Is done by great great grandson, Gary Trudeau. Trudeau inst, I go, is in upstate NY. ELT grad from col med school, 1868, 300$ med school fee, 5$ matric fee. brother died of consumption then. Used to be called consumption; when someone is consumed, is wasting away as if something is eating you...coughing up blood, lose weight, night sweats, characteristic Tb...if you go back 100 years ago, people died primarily of Tb...a leading cause of death back then. It was not uncommon that the primary infectious cause of death, disease 100 years ago was Tb. He watched his brother die in 1868 of Tb, he had been treating his brother, and 3 years later he was diagnosed with Tb. Back then, because you started to cough up blood, you were doomed to die. In 1871, 63rd street, Dr. Janeway's office...was like being sent to the gallows. he fig that if he was going to die, he should do what he enjoyed-->Adirondacks near Lake Placid, Syranack Lake. Chiara: I've been to his house! Bill: Paul smith had a camp where hunters, fishers would go and stay...little red cabin where Tb was treated. We;ll talk in a second. Paul smith college, nearby, teaches hotel management, etc...where Trudeau stayed. Absolutely beatiful in summer, ELT survived winter, summer, and he thought something with the fresh air and sunlight, with rest, saved him. Tb treatment for next 75 years in u.s. was the cure cottage, where treatment was where patient would sit in sunlight, bedrest. All sanitoria started in u.s., trudeau started in saranac lake. if you look across aecom street at van edton hospital, you'll see porches on windows, on rooms, outside. built as a sanitoria to treat Tb patients, where treatment was for coming out of patients in 1953 for outdoor rest. then, first drugs had been discovered and it revolutionized treatment. INH drug then, still used as principle drug. Was built as sanitoria, now used for HIV treatment center, and many of these do have Tb. Reason I love trudeau, is that in his autobiography, this was treatment for Tb, and if you had Tb...lots of famous people had Tb then. Robert Lewis Stevenson, Treasure Island, was there, as a patient. I can imagine ELT and Livingston discussing Tb...1880...picture yourself at that time, and someone asks you, what causes Tb? What would you think? What could the possible causes of Tb be then? Genevieve: malnutrition, wasn't it industrial times, so air pol, crowding? Bill: people wondered. Chiara: Satin. Bill's next slide: wrath of god, inherited trait, vampires, bad air, bacilli.
VAmpires was my favorite hypothesis of then, believed because of the slow wasting, as if being fed on nightly. Takes a long time to die. Who would die next would be the people in your house, so vampire new where to come back. Air, sunlight helped...anti-vampire. The definitive proof was, though, was when dug up body to put stake in heart, would be blood in esophogus. Forget that you cough up blood when have Tb. Point is, the idea that a bacteria was doing this, was obsurd. Reason why we do basic research is that to approach basic research, is that therapies are radically different. Repentence, sterlization, stake in heart, fresh air, ????.
You have to know what you are fighting. Why it is so important to know what causes SARS, HIV...when fighting a war, you have to know your enemy. 1882, Trudea...he got a very spec XMAS present...a full translation of Koch's paper in English...a big hand written copy book. ELT: "One of the greatest, if not the greatest, medical papers ever written and a model of logic of the new experimental method to the study of disease." He was so excited, he set up one of 1st US research labs to duplicate Koch's work. Here he is in his lab, working in Saranac lake, quite different than our biosafety level 3 labs. Everybody that worked there had Tb anyway, not like they wouldn't get it.
I'm almost done for today, but I want to come acrross what was in that paper in 1882. Translating that paper answers the question, how do we know what causes a disease? R. Koch wrote, to prove that Tb is caused by invasion and multipliation of bacilli, it was necessary to:
1) isolate the bacilli,
2) grow it in pure culture,
3) demonstrate that the administration of the same bacilli to an animal reproduces the same morbid conditions.
You know how you are cloning your phages, from asingle plaque? Is what he said about bacilli...stsarting from a single cell colony, and put back into an animal and show it causes disease. isolate bacteria, purify, and put it back. then you know Tb is caused by this bacterium. This is called Koch's postulate, and is directly translated from his 1882 paper.
you know how I was telling you the reason I love genetics, before? The reason we didn't know much about Tb was that we couldn't fulfill Koch's corilary. If you wanted to prove that a phenotype...if you want ...what is a phenotype? Characteristic. appearance, drug resistance, hair color, texture of hair, a property, unique characteristic. if you want to prove that a pheno type...when I see smeg and smell, and know it is smeg, I look at smeg...your phages have sp phenotypes, they form characteristic plaques. If you want to prove that ...very few of your pahgese look alike, plaques. you can describe your phenotype for your phage. you will see that when we do em's next week that they look different, and when we do sequences, you will note that they will all have DNA.
We will prove that they are different next week. If you want to prove that a phenotype is ... you can isolate a phage mutant. You can get a mutation in a gene, of that phage, and a single mutation will make this totally clear. A mutant is just like a disease state, if you think about it. What is a disease? Whatever normal is, a mutant might be a clear plaque, or a tiny plaque. Much of what we are interested in are avirulent mutants; Tb causes disease in animals. Then you have to clone it, clone the genotype. Let's say you are a drug resistant mutant, you have to get a piece of DNA and isolate it. Then you have to transfer it back, and show that it causes an altered phenotype. Over the next couple weeks, we will talk about these simple ideas. Lot's of things will be involved in this. It is how we actually know things. We will fulfill these criteria...we know that if an organism causes a disease, does this all make sense? Listen, tomorrow..we meet here at 10 AM...meet people in the city at 81st street...
Questions answered:
Reason's most leprosy patients die, is they cut themselves, get a secondary infection, and with bandages, can't even feel. Leprosy grows into neurons...ever had fingers anesthetized, i.e. novacaine, this is how leprosy patients are.
Chiara: once a mutant is made, is it possible to know what you mutated to switch it back? Bill, yep, I spent 20 years on this...we'll show you exactly this; must be done to prove this. Chiara: possible to prove? Bill: before, this couldn't be done...our lab has developed these methods. You think thta something asimportnat as Tb, ...please tell mom and dad that you aren't working with Tb, youare working with Smeg. Upstairs, with Tb, we work in a regulated setting, hepa filters everywhere. Division time of Tb slow. 4-6 weeks to get good plates. I never knew how bad my aspetic technique wsa until Tb work. You know how see contamination...with Tb, we have to keep plates 4-5 weeks...slow bacteria show up at 14 days, so fast comparing to Tb. Also, the potato chip effects on plates. Dry out. Also, there were no genetic tools to work with Tb, was that years ago, in my backyard, i isolated bxb1 phage...the bronx bomber, forms big plaques...halo's ; bullseye with halos, but continues to grow. phages have been key for us to develop tools for mycobacteria. we have people in lab from india, africa, mexico, etc... I told you about firefly phages before, and people come to learn these techniques. I may not see you after Aug 1, I'll be in on aug 11 before Africa to wrap up with you...I won't leave until that night. I'm going to Johannsburg, S.A...people come to lab all the time to learn how to use this luciferace phage...like how Reid watches over you, I have others watch over other people. People come in, and someone is going...Bronx Zoo trip stated before was explained...zebra pit. "I smell a phage, put into my pocket, no tube, then. Was BxZ1, first Bronx Zoo from there. Bxz2 from Monkey pit. [[longer tail on Bxz2 than Bxz1...comparing]]
My twin sister, Latrobe penn, teaches HS bio, now we have kids isolating phages from all over the US. ... various phages shown
my sister, a med technologist, did autopsies, then blood banking, then got teaching degree, called me up one day and said I need projects for my kids. Jake and others, got rosebush and barnyard, student changed name to phagehunter at aol.com, is jake. he got rosebush from a rosebush. marvalous collab at Univ Pitt, Graham hatfull, we've collaborated for 12 years. he's a phage sequence guru, marissa is the one who puts this together, she runs the hs course at pittsburg, is a world champion judo expert...runs the sequencing facility. In the back, we will talk more about DNA sequences, etc.
the real trick is to use phages to do cool things...Einstein quote: "Imagination is more important that knowledge." Turn on the light assay, glowing Tb shown. As a geneticist, who is interested in gene transfer, moving a genotype from 1 strain to another, usually involves a cloning step. Listed lengthy slide, "Progress i Mycobacterial Genetics." Good question [chiara], gene transfer. We'll talk more about how we will do simple things in the next
Our discussion: how do we feel about using collectively found phage?